Microbial anabolic and catabolic utilization of hydrocarbons in deep subseafloor sediments of Guaymas Basin

Abstract Guaymas Basin, located in the Gulf of California, is a hydrothermally active marginal basin. Due to steep geothermal gradients and localized heating by sill intrusions, microbial substrates like short-chain fatty acids and hydrocarbons are abiotically produced from sedimentary organic matter at comparatively shallow depths. We analyzed the effect of hydrocarbons on uptake of hydrocarbons by microorganisms via nano-scale secondary ion mass spectrometry (NanoSIMS) and microbial sulfate reduction rates (SRR), using samples from two drill sites sampled by IODP Expedition 385 (U1545C and U1546D). These sites are in close proximity of each other (ca. 1 km) and have very similar sedimentology. Site U1546D experienced the intrusion of a sill that has since then thermally equilibrated with the surrounding sediment. Both sites currently have an identical geothermal gradient, despite their different thermal history. The localized heating by the sill led to thermal cracking of sedimentary organic matter and formation of potentially bioavailable organic substrates. There were low levels of hydrocarbon and nitrogen uptake in some samples from both sites, mostly in surficial samples. Hydrocarbon and methane additions stimulated SRR in near-seafloor samples from Site U1545C, while samples from Site U1546D reacted positively only on methane. Our data indicate the potential of microorganisms to metabolize hydrocarbons even in the deep subsurface of Guaymas Basin.


Introduction
The deep subseafloor biosphere harbors vast amounts of prokaryotes, their number is thought to be a ppr oximatel y the same as in soil and seaw ater (Kallmey er et al. 2012 ).In addition, microorganisms in deep subsurface sediments are metabolically active (D'Hondt et al. 2004, Schippers et al. 2005 ) or at least r e viv able (Morono et al. 2011, 2020, Trembath-Reichert et al. 2017 ).Because of its great amounts of biomass, the deep biosphere is considered to play a vital role in the global cycling of elements (Parkes et al. 2014 ).
Guaymas Basin, located in the Gulf of California (Mexico), is c har acterized by strong hydrothermal activity due to seafloor spreading (Kawka and Simoneit 1987 ).Due to high productivity in its surface waters and in parts high terrigenous sediment input, or ganic-ric h sediment accum ulates at r ates exceeding 1 mm year −1 (Calv ert 1966, Curr ay et al. 1979, Teske et al. 2021a ).In areas with steep geothermal gradients like Guaymas Basin or Nankai Trough off Japan, bioavailable organic substrates like volatile fatty acids are produced by geothermal degradation (pyrolysis) of macromolecular sedimentary organic matter (kerogen) already at shallow depths (Kawka and Simoneit 1994, Horsfield et al. 2006, Teske et al. 2014 ).In addition, laboratory experiments sho w ed that acetate is produced when heating sediment to temperatures in the mesophilic to thermophilic range (Wellsbury et al. 1997 ).These findings support the notion that in situ production of organic substrates can support life in the deep subsurface biosphere .T he supply of carbon sources in Guaymas Basin is therefore expected to be r elativ el y high and div erse (r e vie w in Edgcomb et al. 2022 ).
Recent technological developments , e .g. deep drilling with contamination control for recovery of samples suitable for microbiological and molecular biological analyses, as well as sensitiv e tec hniques e.g. for detections of ultra-low abundances of microbial cells, made it possible to study microbial communities and metabolic activities in the deep biosphere (Colwell and D'Hondt 2013, Morono and Inagaki 2016, Kallmeyer 2017, Morono 2023 ).Additionally, to elucidate the metabolic activity of microorganisms in various en vironments , many molecular biological or c hemical anal ytical tec hniques ar e used.Nano-scale secondary ion mass spectrometry (NanoSIMS) is a po w erful tool to determine solid surface compositions (e.g.minerals and cells) on the singlecell le v el because it is ca pable of nm-scale r esolution (e.g.Ito and Messenger 2008, Kubota et al. 2014, Morono et al. 2020 ).This technique, ther efor e, allows quantification of uptake of stable-isotope labeled substrates at the single-cell level (Lechene et al. 2006, Wagner 2009 ).Recent studies used NanoSIMS to detect viable cells in 20 million-year-old coal bed sediment from depths of over 2000 meters below seafloor (mbsf) off the Shimokita Peninsula, Japan, and in 100 million-year-old sediment of the oligotrophic South Pacific Gyre (Trembath-Reichert et al. 2017, Morono et al. 2020 ).Both envir onments ar e c har acterized by extr emel y low cell abundances (10 4 -10 0 cells cm −3 below 1500 mbsf and 10 6 -10 2 cells cm −3 down to 100 mbsf, r espectiv el y) (Ina gaki et al. 2015, Tr embath-Reic hert et al. 2017, Morono et al. 2020 ).
In anoxic subsurface sediment, after other thermodynamically mor e efficient electr on acceptors (O 2 , NO 3 − , Mn(IV), and Fe(III)) are depleted, sulfate reduction becomes the quantitatively dominant organic matter mineralization process (Jørgensen 1982, 2000, Parkes et al. 2014 ).The biodegradation of hydrocarbons under anaerobic conditions and the respective metabolic strategies have already been studied extensiv el y (e.g.Mec kenstoc k and Mouttaki ( 2011 ) and r efer ences ther ein).Sulfate r educers can also metabolize a wide variety of carbon sources including aliphatic or aromatic hydrocarbons (Reuter et al. 1994, Coates et al. 1996, Shin et al. 2019 ).Sulfate reduction fueled by organic matter is commonly termed organoclastic sulfate reduction.Below the zone of organoclastic sulfate reduction, sulfate reduction can be coupled with methane oxidation (methanotrophic sulfate reduction, or anaerobic oxidation of methane; AOM) through a consortium of archaeal methanotrophs and sulfate-reducing bacteria (Iversen and Jorgensen 1985, Hoehler et al. 1994, Boetius et al. 2000 ).This process is usually restricted to the relatively narrow depth interval where do wnw ar d diffusing sulfate and upw ar d diffusing methane overlap, the so-called sulfate-methane transition zone (SMTZ).Sulfate is not fully depleted in the SMTZ and remains at low μM levels due to the reoxidation of sulfide via a cryptic ir on-driv en sulfur cycle (Holmkvist et al. 2011 ).
Pyr ol ysis of sedimentary organic matter leads to the formation and release of a wide range of hydrocarbons in Guaymas Basin that fuel microbial activity in these sediments (Teske et al. 2014 ).While the microbiology of Guaymas Basin's surface sediments have been studied for decades, the potential of anabolic and catabolic metabolisms by micr oor ganisms living in the deep subsurface of this ecosystem has not been studied due to a lack of suitable samples.Our study aims to elucidate anaerobic microbial metabolic activities involving hydrocarbons in deep subseafloor sediment of Guaymas Basin by addressing the following questions: (1) which types of hydrocarbons are assimilated by microorganisms and (2) do hydrocarbons influence microbial catabolic activity, i.e. is microbial sulfate reduction coupled to hydrocarbon degradation?The first question is addressed through the detection of uptake of stable isotope-labeled aliphatic/aromatic hydrocarbons as well as methane, using NanoSIMS.To address the second question, we quantified the effect of aliphatic/aromatic hydrocarbon or methane addition on microbial sulfate reduction via incubation experiments using 35 SO 4 2 − r adiotr acer.Most hydr ocarbons used for these experiments ( n -decane , n -hexadecane , nicosane , naphthalene , anthracene , phenanthrene , and methane) were detected in Guaymas Basin sediments, the other two hydrocarbons (squalene and benzene) were not measured but expected to exist (Edgcomb, personal communication).

Sampling
The samples wer e r ecov er ed in 2019 during the IODP Expedition 385; Guaymas Basin Tectonics and Biosphere (Teske et al. 2021a , Na gakur a et al. 2022 ).Immediately after sampling, the drilled whole r ound cor e samples wer e placed in nitr ogen-filled gas-tight bags and stored at 4 • C until further subsampling in the home lab.
For our study, we used cores from U1545C and U1546D (Table 1 ).These two sites are about 1.1 km a part fr om eac h other.Str atigr aphy and sediment composition are almost identical at both sites, but sediments at Site U1546D were affected by a sill intrusion.
(From this section, Site U1545C is called "the nonsill site" and Site U1546D is called "the sill site".)Ho w e v er, since the temper atur e gradients at both sites are almost identical (225 • C km −1 at the nonsill site and 221 • C km −1 at the sill site), we can assume that heat from the sill has already dissipated (Teske et al. 2021a, Nagakura et al. 2022 ).Table 1 shows the depth and temper atur e data of the core samples used for this study.The samples were selected from a wide range of temperatures (4

Organic geochemical analyses of hydrocarbons in subsurface sediments
In order to obtain additional information on the geochemical habitat in which the microbial cells are living, and to see whether the natur al hydr ocarbon compositions differ between sites, we analyzed the aliphatic organic compounds using gas c hr omatogr a phy-mass spectr ometry (GC-MS) and nitr ogen, sulfur, and oxygen (NSO) containing compounds using Fourier tr ansform-ion cyclotr on r esonance-mass spectr ometry (FT-ICR-MS).
The aliphatic fraction was measured on a Trace Gas Chromatogr a ph 1310 (Thermo Scientific) coupled to a TSQ 9000 mass spectrometer (Thermo Scientific).The gas c hr omatogr a ph was equipped with a cold injection system operating in splitless mode and a SGE BPX 5 fused-silica capillary column (50 m length, 0.22 mm ID, and 0.25 μm film thickness) using the following temper atur e conditions: initial temper atur e 50 • C (1 min isothermal), heating rate 3 • C min −1 to 310 • C, held isothermally for 30 min.Helium was used as carrier gas with a constant flow of 1 ml min −1 .The injector temper atur e was pr ogr ammed fr om 50 • C to 300 • C at a rate of 10 • C s −1 .The MS operated in the electron impact mode at 70 eV.Full-scan mass spectra were recorded from m/z 50 to 650 at a scan rate of 1.5 scans s −1 .
The NSO fraction was measured in methanol and toluene (1:1, v/v) at a concentration of 25 μg ml −1 negative ion electrospray ionization (ESI) mode using a 12 Tesla FT-ICR-MS solariX upgraded with a P ar aCell equipped with an Apollo II ESI source (both from Bruker Daltonik GmbH, Br emen).Nitr ogen was used as drying gas Table 1.Depth and temper atur e data of the samples from the IODP Expedition 385 nonsill site (U1545C) and sill site (U1546D).The SMTZ at the nonsill site and the sill site are around 40 mbsf and 110 mbsf, respectively.The incubation temperatures were within ±2 at a flow rate of 4.0 l min −1 and a temperature of 220 • C and as nebulizing gas with 1.4 bar.The sample solutions were infused at a flow rate of 150 μl h −1 .The ca pillary volta ge was set to 3000 V and an additional collision-induced dissociation voltage of 60 V in the sour ce w as applied to av oid cluster and adduct formation.Ions wer e accum ulated in the collision cell for 0.05 s and tr ansferr ed to the ICR cell within 1 ms.Spectra were recorded in broadband mode using 8 megaw or d data sets.For each mass spectrum, 200 scans were accumulated in a mass range from m/z 147 to 1000.Sine-bell apodization was applied prior to the Fourier transformation to produce the frequency domain data, which was then converted to the mass spectrum.
Internal quadr atic r ecalibr ation was performed with a standard deviation error < 0.009.Signals with signal-to-noise ratio ≥6 were included in data assessment.Formula assignment was done using the elemental ranges C 5-100 H 5-200 N 0-2 O 0-10 S 0-2 Na 0-1 using a combination of Bruker Anal ysis, Micr osoft Excel, and R. Venn analysis was done in R.

Sample prepar a tion for stable-/r adioisotope experiments
The following pr ocedur es wer e a pplied to both the r adioisotope and stable-isotope experiments described in the following sections.All materials in contact with the sample were either autoclaved or combusted (400 • C for 4 h).All sample handling was carried out inside a nitrogen-filled anoxic glovebox.In the anoxic glovebox, 10 g of sediment was placed into the precombusted glass crimp vial (volume: 30 ml) at ca. 6 • C and mixed with anoxic artificial seawater medium to form a slurry.Note that the outer sediment in whole r ound cor es was not used to avoid contamination.The vial was closed with a thick black butyl rubber stopper without any headspace.For the incubation at approximate in situ pr essur e (ca. 25 MPa) and temperature (Table 1 ), we placed the vials in stainless steel high-pr essur e cylinders.Because of the low compressibility of water, the flexibility of the rubber stopper was enough to transfer the pressure of the high-pressure cylinder into the vial.Since we realized that the thick rubber stoppers did not work pr operl y to tr ansfer the pr essur e at low temper atur es, we cut off the bottom 5 mm of the stoppers to make them thinner, and hence more flexible.

Quantification of hydrocarbon and inorganic nitrogen uptake via NanoSIMS
We aimed to observe hydrocarbon and inorganic nitrogen uptake by incubating sediment samples with stable-isotope-labeled hydrocarbons and ammonium chloride and analyze them using NanoSIMS.

Medium preparation for stable-isotope analyses
The composition of the medium was the same as in Na gakur a et al. ( 2022), but slightly modified for the stable isotope uptake analysis with; 0.2 g KH 2 PO 4 , 0.225 g 14 NH 4 Cl, 0.025 g 15 NH 4 Cl (for nitr ogen uptake anal ysis), 25 g NaCl, 0.5 g MgCl 2 × 6H 2 O, 0.5 g KCl, 0.15 g CaCl 2 × 2H 2 O, and 0.71 g Na 2 SO 4 mixed with 1 l of MilliQ water.3 ml of 0.1% resazurin was added to the medium and autoclaved.5 ml of Na 2 S solution (0.12 g Na 2 S in 10 ml MilliQ water) and 5 ml of NaHCO 3 solution (0.84 g NaHCO 3 in 10 ml MilliQ w ater) w ere added to the medium and flushed with N 2 /CO 2 gas for ca. 2 h.The medium was then stored in autoclaved serum bottles flushed with N 2 /CO 2 gas until use.

Sample incubation with stable isotope substrates
Similar to the SRR measurements (see belo w), w e separated the samples for quantification of anabolic acti vity (i.e.uptak e) via NanoSIMS into two groups, (a) uptake of benzene (C 6 H 6 ) and nhexadecane (C 16 H 34 ) and (b) uptake of methane.Both types of samples were amended with 15 N-ammonium chloride to monitor uptake of inorganic nitrogen.(a) Each 10% (w/v of acetone) hydrocarbon stock solution ( n -hexadecane and benzene) was prepared after Widdel and Bak ( 1992 ).The n -hexadecane stock solution consisted of 90wt% of n -hexadecane and 10wt% of fully deuterated nhexadecane-d 34 .The benzene stock solution consisted of 80wt% of benzene and 20wt% of benzene-13 C 6 .When the medium was added to the glass vial containing the sample, 400 μl of each hydr ocarbon stoc k solution was added as well, and the sample vial was closed with a black butyl rubber stopper and crimped.The stoc k solutions wer e added to the sample vials at r oom temper ature to k ee p the hydr ocarbons dissolv ed in acetone.In addition, a 3-ml syringe including 0.5 ml of the medium was placed into each vial to avoid br eaka ge of the glass vial upon pressurization.The samples wer e pr eincubated ov ernight in the anoxic glov ebox to allow the micr oor ganisms to adjust to the new conditions.(b) For the incubation with methane, the sediment samples were mixed with the medium and preincubated in the anoxic glovebox overnight.After preincubation, 10 ml of gaseous methane, consisting of 80v% 12 C-methane and 20v% of 13 C-methane was injected into the sample vial.For both types of incubations we also pr epar ed two killed contr ols (KCs), whic h wer e mixed with 20% ZnAc instead of media.Other conditions were the same as the samples mentioned above; one KC was prepared with hydrocarbons (a), and the other one with methane (b).Since there is no microbial activity in KCs, the labeled substr ates ar e not incor por ated and can be used for the statistical criterion (see below).
The samples were put into high-pressure cylinders and they were incubated in a HPTGB system (Kallmeyer et al. 2003, Nagakura et al. 2022 ) at in situ temper atur e (Table 1 ) and pr essur e (ca. 25 MPa) for 42 da ys .

Sample fixation
At the end of the incubation, samples were fixed in phosphatebuffered saline (PBS) solution and paraformaldehyde (PFA) solution.The solutions were prepared as 10x PBS and 9.33% PFA.For 10x PBS solution, 79.4 g NaCl, 1,9 g KCl, 11.4 g Na 2 HPO 4 , and 2.6 g KH 2 PO 4 were mixed in 1 l MilliQ (pH 7.2).The solution was then autoclaved and stored at room temperature.For 9.33% PFA solution, 28 g of PFA was added to 260 ml MilliQ water and heated to ca. 60 • C. As PFA onl y dissolv es in alkaline conditions, 1 N NaOH was added until the PFA was dissolved.The solution was then cooled down and 30 ml of 10x PBS was added.HCl was added to adjust the pH to 7.2.To maintain consistent osmotic conditions, we also added 5.1 g NaCl.The solution was brought to its final volume of 300 ml by adding MilliQ water, then the solution was sterile filtered (0.2 μm pore size).When the PFA solution is mixed with the incubation media, it has a final concentration of ca.4%.
Upon r emov al of the samples fr om the HPTGB, the sample vials were opened and the sediment and medium wer e immediatel y tr ansferr ed into 50 ml centrifuge tubes including 15 ml of 9.33% PFA solution and stored for 22 h at 4 • C.After this fixation step, the samples were centrifuged at 2500 ×g for 15 min and the supernatant was discarded.Afterwards, the samples were washed twice with 1x PBS.Once the samples were fixed, they were preserved in PBS/ethanol (1:1, v/v) and k e pt at −20 • C until analysis at JAMSTEC in Koc hi, Ja pan.

Cell detaching and sorting
At JAMSTEC, the following pr ocedur es (modified from Morono et al. 2013 ) were performed for detaching cells from sediments.
(1) 0.5 ml of the sample slurries wer e tr ansferr ed into new 15 ml centrifuge tubes and mixed with 1 ml of 2.5% NaCl.
(2) Samples were centrifuged at 5000 × g for 10 min and the supernatants were discarded.
(7) The supernatants were collected and tr ansferr ed into ne w 15 ml centrifuge tubes.(8) 5 ml of NaCl was added to the sediment and resuspended.(9) The slurry was centrifuged at 6000 × g for 15 min and the supernatant was discarded.(10) The rest was mixed with 2.2 ml of NaCl, 0.4 ml of D-mix, and 0.4 ml of methanol.(11) The mixed solution was shaken at 500 r/m for 10 min.(12) The sample was then sonicated for 20 min.(13) The sample was placed gently onto the gradient layer solution in 15 ml centrifuge tubes.( 14) The tube was centrifuged at 10 000 × g for 1 h.(15) The supernatant was car efull y r ecov er ed and stor ed with the supernatant collected before.
The cells in the supernatant were collected by filtration through an Anodisc TM 25 alumin um o xide filter (pore size 0.2 μm).500 μl of 1x TE buffer was also placed on Anodisc to wash out the density-gradient compounds.After removing the solution and stopping the vacuum pump, 110 μl of 40x diluted SYBR Green I (Thermo Fischer Scientific) was immediately put onto the filter and left for 10 min.After 10 min, 500 μl of 1x TE buffer was poured onto the Anodisc membrane while vacuuming to wash the membrane.Right after the final drop of the liquid had passed through the membrane, the membrane was immediately put in a 50-ml centrifuge tube containing 5 ml of 1x TE buffer, placing the side containing the cells facing down.The tube containing the filter was then sonicated twice for 30 s each at 200 W. Then the suspension was stored at 4 • C.
The stained cells were sorted by a cell sorter (MoFlo XDP, Beckman Coulter).Cells were directly sorted onto an indium tin oxide (IT O)-coated membrane .Approximately, 10 000 cells were sorted on the IT O membrane .For the sample from the nonsill site Core 16 incubated with benzene and n -hexadecane, 30 000 cells were sorted.The area where the cells were sorted was marked by laser microdissection (LMD6000; Leica Microsystems).

Analysis of h ydr ocarbon uptake with NanoSIMS
The sorted microbial cells on the ITO-coated membrane (Morono et al. 2020 ) wer e anal yzed with the JAMSTEC NanoSIMS 50L ion micr opr obe (AMETEK Co. Ltd, C AMEC A BU).In the samples , incubated with benzene and n -hexadecane, 1 H − and 2 H − were analyzed after the analysis of carbon and nitrogen isotopes .T he anal ytical pr ocedur es wer e described else wher e (e.g.Mor ono et al. 2020 ).In brief, a focused primary positive Cs ion beam of ∼1.5 pA was used for carbon and nitrogen isotopic analyses, and approximately ∼6 pA was used for hydrogen isotopic analysis, rastered over 24 μm × 24 μm areas on the samples.Each analysis was initiated after stabilization of the secondary ion beam intensity following se v er al minutes of pr esputtering with a r elativ el y str ong primary ion beam current ( ∼20 pA).For carbon and nitrogen isotopic anal ysis, ima ges of 12 C − , 13 C − , 16 O − , 12 C 14 N − , 12 C 15 N − , and 32 S − wer e acquir ed sim ultaneousl y in m ultidetection with six electr on m ultipliers (EMs) at a mass r esolving po w er of ∼9000, sufficient to separate all relevant isobaric interferences ( 12 C 1 H on 13 C and 13 C 14 N on 12 C 15 N).F or h ydr ogen isotopic anal ysis, ima ges of 1 H − , 2 H − , and 12 C − wer e acquir ed using thr ee EMs in m ultidetection mode at a mass resolving po w er of ∼3000.Eac h anal ysis consisted of the same ar ea, whic h individual images consisting of 256 pixels × 256 pixels .T he dwell times were 2 ms pixel −1 (131.072s scan −1 ) for the carbon and nitrogen isotopic analyses and 5 ms pixel −1 (327.68 s scan −1 ) for the hydrogen isotopic analysis.

Examination of isotope abundance ratio with OpenMIMS
After the NanoSIMS analysis was performed, the data was saved in IM files.To open and c hec k the NanoSIMS ima ges, the Open-MIMS plugin in the application ImageJ of Fiji software was used.Each of the vertically stacked spattered planes was aligned to correct the drift during the acquisition of each plane image and integrated into an image.
To identify the cellular regions, the NanoSIMS images were compared to the fluorescence microscopy images, which were taken before (Fig. 1 ).We were able to match the isotope images and the fluorescence microscopy image in about half of the samples.
Cells shown in the isotope ima ges wer e marked as regions of interest (ROI) and the isotope abundance of each R OI w as thereb y calculated. 13C/( 12 C + 13 C), 12 C 15 N/( 12 C 14 N + 12 C 15 N), or 2 H/( 1 H + 2 H) isotope abundance ratios were calculated to examine if the cells assimilated the isotopes.R OI w er e dr awn based on the following criteria: (1) R OI w er e dr awn based on the clear isotope signals in 12 C images.When the signals are clearly detected, R OI w ere drawn also based on the 13 C images or 12 C/ 15 N images (heavy isotope images).( 2) If at least one of the isotope abundance ratios was zero, the R OI w as excluded.These criteria w er e also a pplied to the samples matching the NanoSIMS and the fluorescence microscopy images.
Since the calculated values are not absolute values but relativ e v alues, the ROI in eac h anal ysis wer e standar dized b y the "blank" ROI drawn at a membrane region without any cells.This blank region is regarded as the natural abundance ratio of each isotope ( 13 C/( 12 C + 13 C) = 1.06%, 15 N/( 14 N + 15 N) = 0.4%, and 2 H/( 1 H + 2 H) = 0.0115%; Trivedi et al. 2016 ).The isotope abundance r atios wer e calculated as follows (her e pr esented for carbon as an example).
Isotope abundance ratio ( C , % ) = 13 C / 12 C + 13 C × 100 .This calculation was also done for the KCs to obtain the av er a ge and standard deviation values for the statistical threshold.
Statistical threshold ( −) = ( Av er a ge of the isotope abundance ratio of KC ) +3 × ( Standard deviation of the isotope abundance ratio of KC ) .This calculation assumes that the ROI values of KCs are normall y distributed.The v alues wer e then conv erted to the isotope abundance ratios and described in percentages.
Since 13 C/( 12 C + 13 C) and 12 C 15 N/( 12 C 14 N + 12 C 15 N) were measur ed separ atel y fr om 2 H/( 1 H + 2 H), w e tried to match the R OI to those based on the carbon, nitrogen, and hydrogen isotope images as m uc h as possible.Based on the position of cells in each isotope ima ge, the cells wer e manuall y matc hed to eac h other and summarized in the 3D plot.

Radioisotope experiment for the measurements of SRR
In order to quantify the effect of hydrocarbon addition on SRR, we incubated the samples with 35

Medium preparation
Medium composition and pr epar ation for SRR measur ements ar e the same as in Na gakur a et al. ( 2022): 0.2 g KH 2 PO 4 , 0.25 g NH 4 Cl, 25 g NaCl, 0.5 g MgCl 2 × 6H 2 O, 0.5 g KCl, 0.15 g CaCl 2 × 2H 2 O, and 0.71 g Na 2 SO 4 were mixed with 1 l of MilliQ water.3 ml of 0.1% resazurin was added to the medium and autoclaved.After autoclaving, 5 ml of Na 2 S solution (0.12 g Na 2 S in 10 ml MilliQ water) and 5 ml of NaHCO 3 solution (0.84 g NaHCO 3 in 10 ml MilliQ) were added to the medium and flushed with N 2 /CO 2 gas for ca. 2 h.The medium was then stored in precombusted crimp bottles flushed with N 2 /CO 2 gas until use.

Sample preparation and incubation with 35S sulfate and hydrocarbon substrates
We quantified SRR with two different hydrocarbon additions, (a) a mixture of eight hydrocarbons and (b) methane.(a) We prepared eight stock solutions, each containing one hydrocarbon in a concentration of 10% (w/v in acetone) (Widdel and Bak 1992 ).The eight stock solutions contained the aliphatic hydrocarbons n -decane (C 10 H 22 ), n -hexadecane (C 16 H 34 ), n -icosane (C 20 H 42 ), squalene (C 30 H 50 ), as well as the aromatic compounds benzene (C 6 H 6 ), naphthalene (C 10 H 8 ), anthracene (C 14 H 10 ), and phenanthrene (C 14 H 10 ).In the ano xic glovebo x, the medium and 100 μl of eac h hydr ocarbon stoc k solution wer e added to the sample vial containing the sediment.The hydrocarbon addition into the sample vial was performed at r oom temper atur e to k ee p hydrocarbons dissolved in acetone .T he sample vial was then closed with a black butyl rubber stopper and crimped.Since n -icosane, anthr acene, and phenanthr ene did not dissolv e completel y in acetone, the y were ad ded as suspensions .T he samples were k e pt in the ano xic glovebo x overnight at ca. 6 • C. (b) For the incubation with methane, the medium was added to the glass vial containing the sample .T he sample vial was closed with a black butyl rubber stopper, crimped, and k e pt in the ano xic glovebo x overnight at ca. 6 • C. The next day, a syringe containing 10 ml of methane was connected to the sample vial with an injection needle through the rubber stopper.All samples were prepared in duplicates, as triplicates were not possible due to limited amounts of sample material.Table 1 shows the incubation temper atur e of eac h sample.Additionall y, eac h run of incubations included KCs and medium controls (MCs).For KCs, sediment was mixed with 20% ZnAc instead of medium and either 100 μl of each hydrocarbon stock solution or 10 ml of methane.MCs contained only media.These contr ols wer e used to confirm that sulfate reduction was carried out biologically and that neither the medium (MCs) nor the sediment (KCs) causes abiotic sulfate reduction, and thus interferes with the quantification of biological turnover.After the preincubation, the samples, as well as the KCs and MCs, were injected with 5 MBq 35 SO 4 2 − r adiotr acer and incubated in our high-pr essur e thermal gr adient bloc k (HPTGB) (Kallmeyer et al. 2003, Na gakur a et al. 2022 ) at in situ temper atur es and pr essur e (ca. 25 MP a) for 10 da ys .In short, the HPTGB consists of a thermally insulated 1.5 mlong aluminum block, the ends of the block can be heated or cooled individually to achieve a thermal gradient.There are three lines of 15 holes each in the bloc k, eac h hole can house one highpr essur e cylinder.Ev ery line has its own pr essur e system, so we can incubate samples at up to 45 different pressure/temperature conditions.After incubation, the high-pr essur e c ylinders w ere depressurized, and the sample vials were removed from the cylinders.Upon opening the glass vials, the samples were immediately poured into 50 ml centrifuge tubes containing 5 ml of 20% ZnAc.
To ensure quantitative transfer of sample and medium, we rinsed the glass vials twice with 5 ml of 20% ZcAc eac h, whic h was added to the same centrifuge tube.Samples were stored at −20 • C until analysis.

Sample distillation and scintillation counting followed by SRR calculation
All inor ganic r educed sulfur species (total r educed inor ganic sulfur, TRIS), which also contain the microbially produced r adiolabeled sulfide, wer e separ ated fr om the sample by cold c hr omium distillation (Kallmeyer et al. 2004 ).After thawing the samples, they were centrifuged for 10 min at 2500 × g .To quantify the total radioactivity, 50 μl of the supernatant was transferred to a scintillation vial and mixed with 4 ml of scintillation cocktail (Rotiszint ® eco plus LSC-Univ ersalcoc ktail, Carl Roth).The remainder of the supernatant was carefully decanted off and the sediment sample was mixed with 15 ml of N , Ndimethylformamide and quantitativ el y tr ansferr ed to a glass distillation flask.A magnetic stir bar was put into the flask and set at 400 r m −1 to ensure complete mixing of the sample and chemicals .T he flask was flushed with N 2 to maintain anoxic conditions.After 10 min of N 2 flushing, 8 ml of 6 M HCl and 15 ml of 1 M c hr omium (II) c hloride solution wer e added thr ough a r eaction port to convert all reduced sulfur species in the sediment sample to gaseous H 2 S. The H 2 S was driven out of the solution by the constant stream of N 2 gas and led through a first trap filled with 7 ml of citric acid solution (19.3 g of citric acid and 4 g of NaOH in 1 l MilliQ water; pH 4) to tr a p all aer osols, potentiall y containing unreacted 35 S-sulfate, before reaching a second trap filled with 7 ml of 20% ZnAc solution in which the H 2 S is quantitatively converted to solid ZnS.To a void o verflowing of the zinc acetate trap, a fe w dr ops of silicon-based antifoam wer e added.The distillation lasted for 2 h.Normall y, onl y 5% ZnAc solution is used for the tr a ps, but the amounts of sulfide in the sample r equir e higher concentrations of ZnAc to ensure the tr a pping of all sulfide.To avoid the possible interference by high concentrations of acetate with the scintillation cocktail, the 20% ZnAc solution was centrifuged at 2500 × g for 10 min and the supernatant was discarded.
The ZnS pellet was resuspended with 5% ZnAc and the total volume was adjusted to 7 ml.The ZnS suspension was then quantitativ el y tr ansferr ed into a 20-ml plastic scintillation vial and mixed with 8 ml of scintillation cocktail.Distillations were carried out in batches of 10 samples plus one distillation blank (DB), containing only a few drops of nonradioactive ZnS carrier.Counter blanks contained only 7 ml of 5% ZnAc solution and 8 ml of the scintillation cocktail.MCs and DBs were then directly transferred into plastic scintillation vials and mixed with 8 ml of scintillation cocktail.
Radioactivity was quantified using a HIDEX 600 SL liquid scintillation counter (HIDEX Oy) with guard scintillator.Before the vials were placed into the counter, they were vortexed to ensure complete mixing of the sample and scintillation cocktail, and the surface of the vial was wiped with a cleaning wipe (Kimtech Science) moistened with 70% ethanol to r emov e an y potential contamination on the surface of the vial.
SRR was calculated as follows: where SRR is calculated in pmol cm −3 d −1 , (SO 4 2 − ) T O T is the total amount of sulfate in the sample (sulfate in the sediment + sulfate in the medium; pmol), V SED is the volume of the sediment sample (cm 3 ), a TRIS is the radioactivity of TRIS (Bq), a T O T is the total used radioactivity (Bq), t is the incubation time (d), and the value 1.06 is the correction factor for the isotopic fractionation of sulfur (Jørgensen 1978 ).Since the samples were incubated with media in a slurry, we consider the results as "potential" SRR.The minimum quantification limit (MQL) and minimum detection limit (MDL) were calculated as follows: MDL = Av er a ge v alue of blank a TRIS ( KCs , MCs , DBc , and CBs ) where k is a factor for a confidence le v el (Kaiser 1970 ).k = 3 was applied for the MQL and its confidence level is 95% instead of 99.86% as the blanks are non-normally distributed (Kaiser 1970 ).The a TRIS of samples and blanks are compared to determine MDL and MQL.

Hydrocarbon compositions in subsurface sediment
GC-MS anal yses r e v ealed that the abundance of n -alkanes differs slightly with depth, but there is no clear difference between the two sites (Fig. 2 ).A homologue series of n -alkanes ranging from n -C 18 to n -C 36 was observed in all samples inv estigated, wher eas nalkanes longer than n -C 31 hardl y occur.The c hr omatogr ams show a bimodal alkane distribution with a dominant maximum around n -C 23 or n -C 24 with no odd ov er e v en carbon number predominance and a smaller maximum at n -C 29 in the long chain range with a distinct odd over even carbon number predominance.Immatur e or ganic biomass contains n -alkanes with a strong odd ov er e v en carbon number pr edominance, whic h gets lost during geothermal maturation (Bray and Evans 1961 ).T hus , the nalkane distribution in the Guaymas Basin sediments indicates that geothermally generated mature hydrocarbons (fossil hydrocarbons; no carbon number predominance) overprint the natural immature n -alkane signal (odd carbon number predominance), which is still visible in the long chain range around the maximum at n -C 29 (Mangelsdorf and Rullkötter 2003 ).The immature n -alkane signal, especially visible in Core 6 samples (Fig. 2 ), represents the typical n -alkane distribution from higher land plants from the adjacent continent (Eglinton and Hamilton 1967 ).Fossil hydrocarbons dominate in particular in the surface near Core 1 at both sites.Additionally, to the aliphatic hydrocarbon fraction the polar compound fraction containing NSO-containing compounds (NSO fr action) wer e anal yzed using FT-ICR-MS.This anal ysis was done, because the free hetero-atomic biomass represents a good substrate source for microbial degradation, due to the energetically higher vulnerability of the functional group in the hydrocarbon structure .T he FT-ICR-MS technique allows to c har acterize the polar compounds by their elemental composition and to order them into specific compound groups according to their heteroatoms and thus , pro viding an insight into the compositional diversity of the biomass.
The compositional data ar e pr esented in Van Kr e v elen diagr ams, in whic h eac h molecular form ula detected is plotted ac-cording to its O/C and H/C ratios (Fig. 3 A).The data have been processed to a Venn analysis before identifying unique and common compounds in the same depths of the nonsill site and the sill site.Common compounds of each depth are plotted as transparent symbols, while unique compounds are shown in solid colors.The data show mainly six different elemental classes indicated by different colors: CHO, CHN , CHON , CHNS , CHOS , and CHONS compounds .T he main elemental class is the CHO compound group, with carboxylic acids r epr esenting the dominant proportion (60%-72% of CHO).Comparing both sites, the sill site sho w ed a higher compositional diversity of unique compounds and with exception of Core 6 of the sill site also a greater number of unique compounds than the same depth of the nonsill site (Fig. 3 B).The CHN compounds (red data points) in the deepest sample of the sill site r epr esent carbazoles .T heir presence as well as other compounds with low H/C values, inferring higher aromaticity, indicate fossil carbon compounds, pr esumabl y migr ated fr om gr eater depth.

Detection of hydrocarbon and inorganic nitrogen uptake with NanoSIMS
We compar ed hydr ocarbon uptake in the incubated samples with those from the KCs to determine if the uptake was statistically significant (Fig. 4 ).The vast majority of the ROI indicates no uptake of carbon compounds (Fig. 4 A and B).Ho w e v er, a fe w ROI indicate low le v els of benzene uptak e (Fig. 4 A) and visible uptak e of carbon from methane (Fig. 4 B).
Nitr ogen fr om ammonium c hloride was assimilated in almost all samples, especially in Core 1 and Core 6 from the sill site in incubations with methane (Fig. 4 C and D).The highest isotope abundance ratio of Core 1 from the sill site incubated with methane r eac hes mor e than 8%.The median isotope abundance r atio of ROI of Core 6 from the sill site incubated with methane shows about 4%.
Since we added benzene and n -hexadecane sim ultaneousl y in the incubations, the uptake of n -hexadecane was observed by deuterium incor por ation (Fig. 4 E).The isotope abundance ratio of the nonsill site a ppar entl y decr eases with depth, and hence with incr easing temper atur e, though onl y compar ativ el y fe w ROI ar e above the statistically significant value.
As uptake of carbon from benzene or methane and nitrogen from ammonium chloride was analyzed simultaneously with NanoSIMS, we c hec k ed if the y were tak en up sim ultaneousl y.At the nonsill site, only one cell out of 177 cells metabolized both methane and ammonium chloride in Core 6, and another cell out of 56 cells metabolized both benzene and ammonium chloride in Core 43.At the sill site, two cells out of 141 cells metabolized both methane and ammonium chloride in Core 6, and one cell out of 112 cells metabolized both benzene and ammonium chloride in Core 16.This data means that the vast majority of cells did not metabolize two labeled compounds sim ultaneousl y.
Ov er all, the r esults show a small but detectable uptake of hydrocarbons (benzene , n -hexadecane , and methane) and inorganic nitrogen (ammonium chloride).
Based on the 13 C/( 12 C + 13 C), 12 C 15 N/( 12 C 14 N + 12 C 15 N), and 2 H/( 1 H + 2 H) v alues fr om the identical R OI, w e summarized the data in a 3D plot (Fig. 5 ).The figure shows only the results from the samples incubated with benzene and n -hexadecane as the samples incubated with methane were not incubated with a 2 Hlabeled substance.
Our results indicate that there is no simultaneous uptake of multiple isotope-labeled compounds , i.e .hydrogen from nhexadecane , carbon from benzene , and nitr ogen fr om ammonium Figur e 2. T he n -alkane distribution of sediment samples from four different depth from the nonsill site (Site U1545C) and the sill site (U1546D) analyzed with GC-MS.The highest peak of each panel is set to 100% of the relative abundance.chloride .T he samples from near the seafloor tend to have higher incor por ation r atios than those fr om deeper cor es (Figs 4 and 5 ).Ther e ar e two r emarkable signals fr om the nonsill site Cor e 1 (Fig. 5 A).One shows a high nitrogen isotope abundance ratio ( ≈ 2.67%) and another shows a high hydrogen isotope abundance ratio ( ≈ 0.0420%).

SRR and the addition of hydrocarbons
To examine the effect of hydrocarbon additions on microbial catabolic activity, we measured SRR in samples from both sites, the nonsill site (U1545C) and the sill site (U1546D), covering a wide depth and temper atur e r ange (fr om 2 mbsf to 261 mbsf; fr om 4 • C to 63 • C) (Fig. 6 ).The study of Na gakur a et al. ( 2022) presented SRR measured on the same samples but without hydrocarbon additions, briefly, SRR were quantifiable whilst no organic substrate was amended and decreased with depth.At the nonsill site, SRR increased upon the addition of methane and the hydrocarbon mixture in the shallo w est sample (2 mbsf, Core 1) (Fig. 6 A).In Cores 6 and 7 near the SMTZ (44 mbsf and 55 mbsf, r espectiv el y), SRR increased upon the addition of methane, representing the increase of methanotrophic sulfate reduction (AOM), but not when adding the hydrocarbon mixture.In samples from below the SMTZ SRR did not increase at the nonsill site adding either methane or hydrocarbons.At the sill site SRR did not increase upon hydrocarbon addition (Fig. 6 B).Ho w e v er, below the SMTZ at 191 mbsf (Core 23), SRR above MQL were observed upon the addition of methane, whereas without any substrate additions SRR fell below MQL.

Rela tion betw een the assimila tion of organic/inorganic matter and dissimilatory sulfate reduction
Our results indicate a greater uptake of deuterated n -hexadecane than 13 C-benzene (Fig. 4 A and E).Ho w e v er, giv en the different detection of the NanoSIMS for each isotope, we cannot draw any conclusions whether n -hexadecane was actually taken up prefer entiall y ov er benzene.Edgcomb et al. ( 2022 ) sho w ed that linear and nonbr anc hed aliphatic hydr ocarbons like n-hexadecane can be degraded more easily by microorganisms than benzene, which is an ar omatic hydr ocarbon, and thus more stable and recalcitrant.
In samples from the sill site Cores 1 and 6, nitrogen (from ammonium chloride) and methane-derived carbon were assimilated (Fig. 4 B and D), but SRR did not increase with the addition of methane (Fig. 6 B).We inter pr et this observ ation as sulfate-r educing micr oor ganisms being activ e but they did not use methane because of the abundance of more favorable organic compounds produced by diagenetic alteration or geothermal matur ation, trigger ed by the sill intrusion in the past (Teske et al. 2014 ), e v en though the heating fr om the sill at the sill site   (B and D).Black solid lines represent the natural incorporation ratios of each isotope (carbon = 1.06%, nitrogen = 0.4%, and hydrogen = 0.0115%, r espectiv el y).Blac k dashed lines r epr esent the statistical threshold values of the uptake of respective isotopes (A, C, and E: carbon = 1.58%, nitrogen = 0.58%, and hydrogen = 0.0255%, r espectiv el y; B and D: carbon = 1.54%, and nitrogen = 0.57%, r espectiv el y).See the method section for more details.A few data are missing due to the breaking of vials during incubation and the lack of additional sediment to repeat the experiments.Note the different ranges of isotope abundance ratios of each element on the y -axes .(Ra w value data are described in Supplementary data S1 -S4 .)has already ceased (Teske et al. 2021a, Na gakur a et al. 2022 ).Other electron acceptors such as NO 3 − , Mn(IV), or Fe(III) could in principle contribute to the assimilation, but these electron acce ptors are de pleted in the sediment and hence not available for microbial metabolisms (LIMS Reports: https://web .iodp.tamu.edu/ LORE/ , last accessed data: 11 April 2024).The significant ammonium uptake suggests that nitrogen source is either limited in these deep subsurface sediments and/or inorganic nitrogen is taken up faster than hydrocarbons .T his observation concurs with the study of Morono et al. ( 2011 ), in which atomic ratios of nitrogen incorporation from 15 N-ammonium were higher than those of carbon incor por ations ( 13 C-glucose , 13 C-acetate , 13 Cpyruvate , 13 C-bicarbonate , and 13 C-methane), Trembath-Reichert et al. ( 2017 ), in which 15 N-ammonium and 15 N-methylamine were incor por ated although 13 C-methanol and 13 C-methylamine uptake was not observed, and Morono et al. ( 2020 ), in which nitrogen incor por ation was gener all y mor e extensiv e than carbon incor poration and could already be observed within the first 21 days of incubation.
In Core 6 sample from the sill site, nitrogen uptake was higher when the sediment was incubated with methane (Fig. 4 D) as compared to an incubation with a mixture of hydrocarbons (benzene and n -hexadecane) (Fig. 4 C).This result might be due to either methane stimulating or benzene/ n -hexadecane suppressing nitrogen uptake.Although the details remain elusive, nitrogen uptake by micr oor ganisms in deep subsurface sediments is well-documented (Morono et al. 2011, 2020, Trembath-Reichert et al. 2017 ).According to the works by Kellermann et al. ( 2012 ),  inorganic carbon (bicarbonate) is taken up pr efer entiall y ov er methane, but methane is used as an energy source , i.e .for catabolic metabolism.
Because microbial sulfate reduction is a catabolic process and mor e exer gonic than anabolic hydr ocarbon uptake, it can be measured in a shorter incubation experiment.Tr embath-Reic hert et al. ( 2017 ), for example, carried out anabolic process studies using anoxic sediments from off Shimokita peninsula, Japan, and incubated those samples for 2.5 years to r eac h a detectable signal of incor por ation, while Glombitza et al. ( 2016 ) incubated sediment from the same drill core for only 10 days to achieve detectable SRR.We also found that 10 days was gener all y sufficient for SRR detection.Potential SRR in Glombitza et al. ( 2016 ) sho w ed lo w er SRR than ours .T his is pr obabl y also because they incubated their samples not at in situ pr essur e but just slightl y abov e ambient conditions ( ∼2 bar).Since pr essur e can be an important factor to enhance microbial metabolic activity, we cannot simply compare the SRR data.But in general, our SRR data sho w ed higher values.Although our samples for incor por ation studies were incubated for only 42 da ys , which was relatively short, even the uptake of hydrocarbons was successfully observed.Ho w ever, to observe a higher degree of assimilation, a longer incubation time (more than 1 year, for instance) would be needed.
Gene expression data from neighboring holes U1545B (nonsill site) and U1546B (sill site) sho w ed differences between sites (Mara et al. 2023 ).Gene expr essions r elated to se v er al ener gy-r elated metabolisms and methanogenesis at Site U1546B are higher than at Site U1545B.In general, Site U1545B sho w ed little gene expression associated with methane cycling and c hemoautotr ophy.Although their data were obtained from the original core samples, whic h ar e differ ent fr om our data after incubation of 10 days (for SRR measurements) or 42 days (for NanoSIMS substrate uptake anal yses), the differ ent gene expr ession le v els pr ovide an explanation for the different responses of metabolic activities in the nonsill site and the sill site .Nonetheless , gene expression levels related to sulfate reduction are very low at both sites (Mara and Edgcomb, personal communication).

Anaerobic degr ada tion of hydrocarbons and sulfate-reducing microorganisms
It has been already known for decades that sulfate-reducing micr oor ganisms can degr ade both aliphatic and ar omatic hydr ocarbons under anaerobic conditions (e.g.Rueter et al. 1994 , Meckenstock andMouttaki 2011 , and references therein) despite their molecular stability.Given the vast literature on this topic, it is safe to assume that sulfate-reducing microorganisms in the Guaymas Basin sediment can degrade the ar omatic hydr ocarbons used in this study: benzene (Edw ar ds and Grbi ć-Gali ć 1992 , Lovley et al. 1995 , Anderson andLovley 2000 ), naphthalene (Zhang and Young 1999, Mec kenstoc k et al. 2000, Musat et al. 2009), phenanthrene (Zhang and Young 1999, Ramsay et al. 2003, Da vido va et al. 2007, Tsai et al. 2009 ), and anthracene (Ramsay et al. 2003 ).Furthermore, the GC-MS data confirm that fossil hydrocarbons are an integral part of the biomolecule diversity in the sediments of Guaymas Basin (Fig. 2 ) most likely introduced by seeps on the seafloor or eroded source rocks during time of sediment deposition.Therefore, we assume that the increase in SRR was induced by one or se v er al of the added aliphatic/ar omatic hydr ocarbons.With the exception of methane, all eight hydrocarbons (four aliphatic and four ar omatic hydr ocarbons) wer e added sim ultaneousl y, so it is not possible to identify which one was used for dissimilatory sulfate reduction.
The samples from the sill site sho w ed a less positive SRR response to the hydrocarbon addition compared to the samples from the nonsill site (Fig. 6 ).This result may be because the sulfate-r educing micr oor ganisms at the sill site did not metabolize as m uc h of the added hydrocarbons as at the nonsill site, although cell abundances between these two sites are similar ( ∼10 9 cells cm −3 near the seafloor to ca. 10 6 cells cm −3 around 175 mbsf) (Teske et al. 2021b , c ).One possible explanation for these findings might be related to the sill emplaced at the sill site and its impact on the deposited organic matter.Upon heating, macromolecular sedimentary organic carbon might have been pyrolyzed and converted into bioavailable organic substrates in the past (Horsfield et al. 2006 ), so the input of heat into the sedimentary system of the sill site (Teske et al. 2014 ), might hav e incr eased the diversity and availability of microbial substrates.Given the overall low microbial activity in these sediments, especially at greater depths, and the heat sterilization of the sediment around the intruded sill as well as the subsequent recolonization of the sediment, the micr obial substr ates that wer e pr oduced by thermal cr ac king might be still available in the sediments, e v en though the heat effect of the sill at the sill site has already ceased.The extent of the thermal aureole and hence the sterilized zone is still unknown, but the TOC data show that a decrease in TOC concentration due to conversion into volatile or at least mobile compounds is restricted to about 24-37 m above and below the sill, respectiv el y (Lizarr alde et al. 2023 ).Ov er the depth r ange cov er ed by our samples (ca.2-260 mbsf at both sites) TOC concentrations scatter ∼1%-2.5% with no visible trends.As our deepest sample was from about 100 m above the sill, it was well above the estimated zones of oil or gas gener ation (Lizarr alde et al. 2023 ), so we assume no direct influence by the heat from the sill.Ho w ever, Lizarralde et al. ( 2023 ) estimated that 3.3 Mt of sedimentary carbon was released by this sill intrusion via thermal cr ac king of the kerogen.The released carbon would be qualitatively different and potentiall y mor e bioav ailable (Horsfield et al. 2006 ).If those thermogenicall y pr oduced bioav ailable carbon sources ar e still pr esent in the sediment, e v en at very low concentrations, they would provide a reservoir of readily bioavailable substrates for the small micr obial comm unity (Mar a et al. 2023 ), and thus the mor e r ecalcitr ant hydr ocarbons added in our experiment were not utilized, and hence did not lead to an increase in SRR.The data obtained from the FT-ICR-MS analyses reveal carbazoles (heterocyclic aromatic compounds consisting of two benzo groups attached to a centr al pyrr ole ring) in the deepest sample of the sill site indicating that this site might have been influenced by migration of hydr ocarbons fr om below.Furthermor e, the sill site r e v eals a mor e diverse composition of organic compounds with different heteroatoms or hetero-atom combinations (Fig. 3 ).T hus , the FT-ICR-MS data organic matter in these subsurface sediments differs qualitativ el y between sites, as they r e v eal differ ent bioav ailability of organic compounds with variable functional groups .T hese results might, ther efor e pr esent a possible explanation for the differences in uptake of isotopically labeled compounds .T he gr eater div ersity and abundance of potentially bioavailable organic compounds at the sill site, ther efor e pr ovide mor e favor able substr ates than the added isotopically labeled compounds.
Comparing our organic geochemistry data (Figs 2 and 3 ), the anabolic data from our NanoSIMS analyses (Figs 4 and 5 ), and SRR data (Fig. 6 ) as well as the pr e viousl y published molecular biological data (Mara et al. 2023 ), we argue that microorganisms can utilize hydr ocarbons catabolicall y and anabolicall y but use them pr efer entiall y for catabolism.

Summary
Microbial anabolic and catabolic utilization of various hydrocarbons in hydr othermall y influenced subsurface sediments of Guaymas Basin were analyzed using incubation with stable-and radioisotope-labeled compounds as well as organic geochemi-cal anal yses.NanoSIMS anal ysis r e v ealed the uptake of carbon from benzene and methane and hydrogen from n -hexadecane by micr oor ganisms in a r elativ el y short incubation time of 42 da ys .Nitr ogen uptake fr om ammonium c hloride was r ecorded especiall y fr om Cor es 1 and 6 at the sill site, incubated together with methane, indicating the presence of active hydrocarbonmetabolizing processes.Radioisotope experiments sho w ed an increase in SRR in samples from near the seafloor at the nonsill site (Site U1545C) upon the addition of hydr ocarbons, wher eas SRR did not increase in most samples from the sill site (Site U1546D).This result may infer that at the sill site more favorable carbon compounds ar e av ailable, pr obabl y linked to the heating effect of a deeper-located sill intrusion, which had been active in the past.Ov er all, despite the low rate of turnover and substrate incorporation in deep subseafloor sediments of Guaymas Basin, our study shows that the indigenous micr oor ganisms can utilize hydrocarbons for both anabolism and catabolism e v en in the deep subsurface of Guaymas Basin.
T.N. is funded through a grant from the Deutsche Forschungsgemeinschaft (DFG) to J.K. (grant number 670521).T.N .' s research tr av el to JAMSTEC was funded by the European Consortium for Ocean Research Drilling (ECORD) Researc h Gr ants 2021 for Early Career Scientists aw ar ded to T.N.

Figure 1 .
Figure 1.NanoSIMS isotope images and fluorescence microscopy images of the sample Core 6 at the sill site (U1546D), incubated with methane.(A) 12 C isotope images analyzed with NanoSIMS.(B) 12 C 14 N isotope images analyzed with NanoSIMS.(C) Isotope ratio image of 13 C/ 12 C, (D) isotope ratio image of 12 C 15 N/ 12 C 14 N, and (E) fluorescence microscopy images .T he cells were dyed with SYBR Green I.The color code with numbers (C and D) indicates the isotope ratios.Scale bars in each panel represent 5 μm.The isotope images were processed with ImageJ.

Figure 3 .
Figure 3. (A) Van Kr e v elen dia gr am of or ganic compounds extr acted fr om four differ ent depths fr om the nonsill site (U1545C) and the sill site (U1546D).The color codes r epr esent the different heteroatomic groups within the organic compounds.Unique compounds of each sample are indicated with nontr anspar ent symbols while shared signals are transparent.(B) Number and elemental class affiliation of unique compounds of each sample.

Figure 4 .
Figure 4. Isotope abundance ratios of each sample.(A and B) Carbon uptake from benzene (A) and methane (B).(C and D) Nitrogen uptake from ammonium chloride.(E) Hydrogen uptake from n -hexadecane.(A, C, and E) Samples incubated with benzene and n -hexadecane.(B and D) Samples incubated with methane.KCs were incubated with 15 N-ammonium chloride plus either with benzene and n -hexadecane (A, C, and E) or with methane (B and D).Black solid lines represent the natural incorporation ratios of each isotope (carbon = 1.06%, nitrogen = 0.4%, and hydrogen = 0.0115%, r espectiv el y).Blac k dashed lines r epr esent the statistical threshold values of the uptake of respective isotopes (A, C, and E: carbon = 1.58%, nitrogen = 0.58%, and hydrogen = 0.0255%, r espectiv el y; B and D: carbon = 1.54%, and nitrogen = 0.57%, r espectiv el y).See the method section for more details.A few data are missing due to the breaking of vials during incubation and the lack of additional sediment to repeat the experiments.Note the different ranges of isotope abundance ratios of each element on the y -axes .(Ra w value data are described in Supplementary data S1 -S4 .)

Figure 6 .
Figure 6.(A) Sulfate reduction rates (SRR) at the nonsill site (U1545C).(B) SRR at the sill site (U1546D).Blue circles show SRR measured from the samples incubated with the hydrocarbon mixture, and red circles show SRR measured from the samples incubated with methane.Black circles show SRR measured from the samples incubated without any substrate addition (Nagakura et al. 2022 ).Solid circles indicate SRR > MQL and open circles indicate SRR < MQL.Gray bars indicate the depth of the respective SMTZ (ca.40 mbsf and ca.110 mbsf, r espectiv el y).A fe w data points ar e missing due to the loss of vials during incubation.Due to the lack of sediment, these experiments could not be repeated.All incubations were carried out at in situ temper atur e and pr essur e .(Ra w v alue data ar e described in Supplementary data S5 .) • C-63 • C) at similar depths of these two sites.Additionally, we chose one sample from each site that is located near the SMTZ.The measured data in this paper is comparable to the pr e vious data (Na gakur a et al. 2022 ).As the measur ements in Na gakur a et al. (2022) were performed as soon as possible after the expedition, the change in microbial community during the sample conservation at 4 • C would be minimal.

Site U1545C (nonsill site) 27 • 38.2420 N 111 • 53.3290 W Site U1546D (sill site) 27 • 37.8943 N 111 • 52.7812 W Core number Depth (mbsf) In situ temper a ture ( • C) Incubation temper a ture for radioisotope experiment ( • C) Incubation temper a ture for stable isotope experiment ( • C) Core number Depth (mbsf) In situ temper a ture ( • C) Incubation temper a ture for radioisotope experiment ( • C) Incubation temper a ture for stable isotope experiment ( • C)
• C of their in situ temper atur es.
SO 4 2 − r adiotr acer and a mixture of v arious hydr ocarbons or methane .T he SRR measurements without hydrocarbon additions were already presented byNagakura  et al. ( 2022 ).